Sars cov 2 plpro

To determine if the inhibitors are Zn-ejecting agents, the presence of free Zn²⁺ in solution was measured as previously described.^{11 (link)} The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mM HEPES, pH 7.5) to 2 μM and 20 μM concentrations. Volumes of 50 μL of 1 μM SARS-CoV-2 PL^pro (Elabscience) in HEPES buffer or blank HEPES buffer (negative control) were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (500 nM PL^pro SARS-CoV-2, 0.5% DMSO, 1 μM or 10 μM test compound or blank HEPES buffer) were mixed. A volume of 100 μL of 2.0 μM of zinc-specific fluorophore FluoZinTM-3 (Invitrogen/Life Technologies) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every 5 min for 100 min (λ_exc = 485 nm; λ_em = 535 nm) at 37 °C using a Victor™ X4 Pekin Elmer 2030 multilabel reader. The wells containing the negative control were used to confirm the absence of false positive results by reaction of the inhibitor compound with the zinc-specific fluorophore.

The inhibitor compounds were prepared as stock solutions in DMSO and diluted hundredfold with HEPES buffer (50 mm HEPES, pH 7.5, 0.1 mg mL⁻¹ bovine serum albumin, 0.1% Triton-X100) to micromolar concentrations. Volumes of 50 μL of 200 nM SARS-CoV-2 PL^pro (Elabscience) in HEPES buffer were added to the wells of a black 96-well microtiter plate (Greiner Bio One). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (positive control) were added. The resulting solutions (100 nm SARS-CoV-2 PL^pro 0.5% DMSO, 0.1–0.75 μm test compound or blank HEPES buffer) were mixed and incubated at 37 °C for 10 min. A volume of 100 μL of 20–8000 μm of substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC, Bachem) was added to all wells. The resulting solutions were mixed and the fluorescence emission was measured immediately every minute for 1 h (λ_exc = 355 nm; λ_em = 460 nm) at 37 °C using a Victor™ X4 Perkin Elmer 2030 multilabel reader. The enzyme activity of PL^pro was represented by the Michaelis Menten equation (eqn (1)) and the K_m and V_max values were calculated with Lineweaver–Burk equation (eqn (2)) where the slope is K_m/V_max, the intersection with the Y-axis corresponds to 1/V_max, and intersection with the X-axis corresponds to −1/K_m.